Infrared spectra and the structure of 1-methyladenine in an argon matrix and solutions

SpectrochimicaActa, Vol. 51A, No. 5, pp. 843-853, 1995

Pergamon 0584-8539(94)00189-8

Copyright © 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0584-8539/95 $9.50 + 0.00

Infrared spectra and the structure of l-methyladenine in an argon matrix and solutions V . B . PIVOVAROV, S. G . STEPANIAN, I. D . REVA, G . G . SHEINA a n d Y u . P. BLAGOI Institute for Low Temperature Physics and Engineering, Ukrainian Academy of Sciences, 47 Lenin Ave., Kharkov 310164, Ukraine

(Received 22 April 1994; in final form 19 August 1994; accepted 22 August 1994) Abstract--The spectral characteristics of the so-called "rare" imine form of methylated nucleotide purine base adenine is investigated. A study is made of 1-methyladenine (1-mAde) having generally the three tautomeric forms: amine, imino-N9H, and imino-N7H. IR spectra of 1-mAde isolated in Ar matrix at 12 K are obtained in the range 4000-400 cm ~. The normal-coordinate analysis is employed to interpret the experimental spectra. It is shown that isolated 1-mAde molecules can only exist as imine. The set of fundamental vibrational frequencies and force constants of the imine tautomer is found. The relative energies of the 1-mAde tautomers are calculated by the SCF MO LCAO method in the AM1 approximation with complete geometry optimization. This method predicts the stability of the tautomers in decreasing order: imine-N9H, imine-N7H, amine. An IR spectroscopic study of solutions of 1-mAde, and also adenine, 9-methyladenine, 1-methyladenosine, 2-aminopyrimidine, 2- and 4-oxopyrimidine in polar solvents in the range 1750-1500 cm-J is carried out. The results show the presence of a considerable amount of the "rare" imine tautomer in water, as well in nonaqueous solutions.

INTRODUCTION

IN THE recent decades IR spectroscopy of solutions and a solid phase has become a common method of investigation of the structure of nucleic acids components. Some new achievements of recent years in this area have been due to a technique of IR spectroscopy of molecules in solidified noble gases [1] applied to investigation of the structure of nucleotide bases. Thus, in cryomatrices, so-called "rare" enol forms of guanine [2-5], cytosine [6-9] as well as isocytosine [10-12], 2-oxopyrimidine [12-14] and 4oxopyrimidine [13, 15, 16] were revealed for the first time. In terms of construction of the spontaneous mutations theory it is of considerable interest to reveal imines, another type of "rare" tautomeric form of nucleic acids bases and their derivatives. Up to now papers have been published reporting studies of the possibility of amino-imine tautomerism of pyrimidine bases. Thus, the iminoform of 3-methylcytosine has been found in low temperature matrices of solidified nitrogen [10] and argon [17] and the iminoform of 1methylisocytosine in nitrogen [10]. Besides, as was mentioned in Ref. [18] the existence of some amount of the cytosine iminoform was proposed by SZCZESNIAKin Ref. [19], and Ref. [17] reported on attempts to reveal it experimentally in argon and neon matrices. The iminoform of 1-methylcytosine in argon matrix was sought by PERSON and co-workers [20]. The authors of Ref. [21] assign several bands of the experimental spectra of cytosine in argon and neon matrix to the presence of a small amount of the cytosine iminoform. LAPINSK! et al. [18] interpret some bands of the spectrum of 5methylcytosine as due to the iminoform of this compound. These assumptions and conclusions are based on the experimentally revealed alteration of IR spectra of the compounds in question induced by UV irradiation of matrices. The authors of Ref. [10], where the characteristic frequencies of the iminogroup were observed in the spectra, did not use UV irradiation of samples. The characteristic frequencies of the iminogroup vibrations of the 5-methylcytosine keto-iminoform were calculated at ab initio level [18]. At present there are no reports of experimental observation or calculation of spectra of imine forms of purine bases. Thus, it is topical, especially for purines, to obtain experimentally direct spectral characteristics of imine tautomers, primarily the characteristic frequencies of NH and C=N stretching vibrations of the iminogroup and also the frequencies of the ring vibrations. Subsequent studies are to ascertain the physical and chemical conditions of the existence of the "rare" imine forms of bases. 843

844

V. B. PIVOVAROVel al.

7

I

Ilb

lla

III

Fig. 1. Structure of 1-methyladenine tautomers. Closed circles mark nitrogen atoms.

W e s t u d i e d 1 - m e t h y l a d e n i n e which m a y a priori be e x p e c t e d to exhibit a s t r o n g shift o f t h e t a u t o m e r e q u i l i b r i u m t o w a r d s t h e i m i n o f o r m in an inert e n v i r o n m e n t . M o l e c u l e s o f 1 - m e t h y l a d e n i n e h a v e two labile p r o t o n s . T h e t o t a l n u m b e r o f p o s s i b l e p r o t o t r o p i c t a u t o m e r s is t h r e e (Fig. 1). S t r u c t u r e s I a n d I I c o r r e s p o n d to i m i n e t a u t o m e r s with t h e s e c o n d p r o t o n l o c a l i z a t i o n at t h e N7 o r N9 a t o m o f the i m i d a z o l e f r a g m e n t , r e s p e c t i v e l y . S t r u c t u r e I I I c o r r e s p o n d s to t h e a m i n e t a u t o m e r with l o c a l i z a t i o n o f t w o p r o t o n s at t h e N10 p o s i t i o n .

EXPERIMENTAL

Measurements of IR spectra of low temperature matrices IR spectra of 1-methyladenine in argon matrices were measured on samples prepared by simultaneous condensation of flows of the gas phase of the compound in question and argon onto low temperature ( T = 17 K) CsI optical substrate. The compound to be studied was evaporated from a Knudsen cell at 225 °C. The matrix gas was 99.99% argon. The molar ratio of 1methyladenine:argon in samples was monitored by means of a low temperature quartz microbalance and was 1:500. The precision of the microbalance was 3%. To prepare a transparent sample, the CsI substrate temperature was maintained at about 17 K. To prevent heating of the matrix by the spectrometer ray the samples were cooled to 12 K before recording spectra. Spectra were registered by means of the setup based on a modified Specord IR 75 spectrometer (Carl Zeiss, Jena), with the resolution of 3 cm-~ in the range 4000-2500 cm -l and 1 cm -~ in the range 2500-400 cm-1; The signal-to-noise ratio was increased by accumulating spectra in various ranges. To prevent effects of atmospheric COz and water vapour on the spectra, the spectrometer was placed in a pressurized housing blown through with dry nitrogen. Control of the evaporation conditions, the optical substrate temperature, recording and processing of spectra were provided by an IBM PC A T coupled to the spectrometer and the cryostat through the CAMAC system. A unique computational package was used for this purpose. Some details of the measurement arrangement which we developed for IR spectroscopy of matrix isolation can be found in Ref. [7]. The utilization of noble gas matrices provides certain advantages over other spectroscopy techniques: • The matrix contains "solid gas", i.e. fixed states of molecules under study characteristic of the gas phase at the evaporation temperature.

IR spectra of 1-methyladenine in argon matrix and solutions

845

• Molecules of compound under study being sufficiently highly diluted in the matrix. There is no interaction between them; the influence of the environment on these molecules is minmized in the inert gas. • The matrix is transparent over the whole range from the far IR to the vacuum UV region, and therefore information on the finest features of the molecular structure can be obtained. These features eliminate the rotational degrees of freedom of molecules of the compound studied, resulting in essential narrowing of spectral lines and making the spectra more informative.

Measurements of IR spectra of solutions IR spectra of 1-methyladenine and model compound in solvents were measured with Specord M82 and UR-20 spectrometers (Carl Zeiss, Jena) in the range 1750-1500 cm-~ with a double-beam registration scheme. The optical length of the demountable cells was 50 mkm, the windows were of CaF2 and the spacers of Teflon. The decrease in the optical length because of the decrease in thickness of the spacers after packing was estimated from the alteration of interference due to windows of emtpy cells; it was within 7%. All measurements in solutions were carried out at 2125°C. The optical density range was from 0.03 to 0.75 optical density unit, as dictated by solubility of the compounds.

Materials We performed measurements on the following compounds without additional purification: 1-methyladenine (Serva, Germany), 9-methyladenine, 1-methyladenosine, adenine (Sigma, U.S.A.), 4-oxopyrimidine (Aldrich, U.S.A.) and 2-oxopyrimidine (Chemapol, Czechoslovakia). We also used 2-aminopyrimidine (Olainen Chemical Reagents Plant, Latvia) which was subjected to double recrystallization from water solution. The compounds were deuterated by dissolving in D20 with subsequent low temperature distillation of D20 vapour. After deuteration the compounds were dried by keeping over P205 powder. The solvents were D20 and CH3OD (Isotope Industrial Association, St. Petersburg), distilled CH3OH and also 1,4-dioxane (Aldrich, U.S.A.) without additional purification.

CALCULATIONS

Relative energies and structure of tautomers The relative energies of tautomers were calculated by the semiempirical quantum chemical method SCF M O L C A O in the AM1 approximation with full geometrical optimization [22]. It was taken into consideration that various isomers could be formed owing to the c/s- and trans-positions of the iminogroup proton and N1 atom.

Normal-coordinate analysis The spectroscopic package [23] was employed to perform the normal-coordinate analysis for the most stable tautomer I (Fig. 1). In the imine tautomer molecule one methyl group hydrogen atom lies in the ring plane representing the symmetry plane and the two other hydrogen atoms lie symmetrically on both sides of this plane. The molecule has Cs symmetry. By the symmetry of the molecular model, normal vibrations of 1-methyladenine were grouped into 32 vibrations belonging to A ' species and 16 vibrations belonging to A" species. All vibrations of 1-methyladenine are IR active. In the capacity of the internal vibrational coordinates of 1-methyladenine (Table 1) we used alterations of all the bond lengths, the bond angles and also the torsion angles. In this case the total number of coordinates introduced is larger than the number of degrees of freedom ( 3 N - 6), owing to appearance of the redundant coordinates. These coordinates were excluded automatically, with use of the algorithms described in Ref. [23]. The coordinates S~-S~9 correspond to alterations of the bond lengths (A r) and the coordinates S20-S49 to alterations of the bond angles (Aa). The out-of-plane coordinates $50, Sst and $53-$55 (Ap) describe alterations of the angles between the respective bonds and the ring plane, and finally the coordinates $52, Ss6-S6s (Ar) describe alterations of the torsion

846

V. B. PIVOVAROVet al. T a b l e 1. I n t e r n a l c o o r d i n a t e s of the 1 - m e t h y l a d e n i n e a No. D e s c r i p t i o n

No. D e s c r i p t i o n

Stretching modes b St = A r N 1 - C 2 $2 = A r C 2 - N 3 $3 = A r N 3 - C 4 $4 = A r C4--C5 $5 = A r C5--C6 $6 = A r C6--N1 $7 = A r C5--N7 $8 = A r N 7 - C 8 $9 = A r C8--N9 S10 = A r N 9 - C 4 Sll = A r C 6 - N 1 0 $12 = A r N10--H1 1 Sl3 = A r N 1 - C 1 2 Si4 = A r C 1 2 - H 1 3 Sis = A r C 1 2 - H 1 4 $16 = A r C 1 2 - H 1 5 SiT = A r C 2 - H 1 6 S18 = A r C8--H17 $19 = A r N 9 - H 1 8

Bending $2o = A a $21 = A a $22 = A a $23 = A a $24 = Act $25 = Act $26 = A a 527 = A a $28 = A a $29= A a $3o = Act $31 = A a $32 = A a $33 = Act $34 = A a $35 = A a $36 = A a $37 = A a $38 = A a $39 = A a $40 = A a S41 = A a $42 = A a $43 = A a $44 = A a $45 = A a $46 = A a 547 = A a $48 = A a $49 = A a

Out-of-plane modes a $50 = A R C 2 - H 1 6 Ssi = A p C6--N10 $52 = A r N 1 - C 6 - N 1 0 - H 1 1 $53 = A p N 1-C12 $54 = A R C 8 - H 1 7 $55 = A p N9--H18

$56= $57 = Ss8= $59 = $6o = S61 = $62 = $63 = $64 = $65 =

modes c N1-C2-N3 C2-N3-C4 N3-C4-C5 C4--C5-C6 C5-C6-N1 C6--N1-C2 C4--C5-N7 C5--N7-C8 N7-CS-N9 C8---N9-C4 N9-C4-C5 N1-C2-H16 N3-C2-H16 N3-C4-N9 C6--C5-N7 C5--C6-N10 N1-C6-N10 C6--N10--H11 N1-C12-H13 N1-C12-H14 N1-C12-H15 H13-C12-H14 H13-C12-H15 H14-C12-H15 N7-C8-H17 N9-CS--H17 CS-N9-H18 C4-N9-H18 C2-N1-C12 C6-N1-C12

Ar C6-N1-C2-N3 At N1-C2-N3-C4 Ar C2-N3-C4-C5 A r N3--C4-C5-C6 Ar C4-C5-C6-N1 At C5-C6-N1-C2 At C4--C5-N7-C8 Ar C5-N7-C8-N9 A1:NT-C8-N9-C4 A r C8--N9-C4-C5

a I n t e r n a l c o o r d i n a t e s for t a u t o m e r 1. b r is the d i s t a n c e b e t w e e n a t o m s . c a is the a n g l e b e t w e e n bonds. dp is the a n g l e b e t w e e n b o n d and ring p l a n e ; r is the torsion angle.

angles. All out-of-plane vibrations are of the A" symmetry type. Besides these, this species includes the one stretching vibration of the methyl group (+S~5, --S16) and also the three bending vibrations. The set of force constants of 1-methyladenine is available from the authors.

RESULTS AND DISCUSSION

Spectrum in an argon matrix As mentioned imine

and

amine

above,

the molecular

tautomers.

An

structure

important

of 1-methyladenine

aim of the present

is such that it forms

investigation

is finding

out

IR spectra of 1-methyladeninein argon matrix and solutions

847

what tautomers of 1-methyladenine exist in an argon matrix. Some preliminary conclusions can be made on the basis of calculated relative energies of tautomers of 1methyladenine which are presented in Table 2. As is seen, the calculations predict preferential stability of imine tautomers I and IIa (Fig. 1). These iminoforms with a proton localized at atom N9 (tautomer I) and at atom N7 (tautomer IIa) of the imidazole fragment have a close value of energy (energy difference A E - - 0 . 7 9 kcal mol-~). Imine tautomer IIa with a proton at atom N7 has a close value of energy (energy difference A E = 0 . 7 9 kcal mol-l). Amine tautomer III is the least stable among the tautomers of 1-methyladenine studied ( A E = 5.44 kcal mol-~). It may be supposed that, when in the isolated state, 1-methyladenine may exist in the iminoform, as structure I and possibly structure lla. The final conclusions on the structure of 1-methyladenine are obtained as a result of analysis of the IR spectrum of the matrix which is presented in Fig. 2 and Table 3. The experimental spectrum was interpreted on the basis of normal-coordinate analysis for the most stable imine tautomer. This was carried out with the use of the spectral information obtained by the matrix isolation method for a variety of related compounds: adenine and 9-methyladenine [24] (amine tautomers), 3-methylcytosine [11] (imine tautomer), and also for purine [24] and imidazole [25]. In order to reveal the imine tautomer, first of all we considered spectral regions containing bands of stretching and bending vibrations of amine and imine groups. N - H modes It is first of all important that the range of N H stretching vibrations (3600-3300 cm- 1) of the spectrum of 1-methyladenine does not contain the bands of antisymmetrical and symmetrical NH2 stretching vibrations which were reliably identified for adenine and 9-methyladenine. These vibrations in matrix spectra of the mentioned compounds appeared as doublets 3564, 3556 cm -1 (NH2 str as) and 3447, 3440 cm -1 (NH2 str s) for adenine, and the 3557cm -1 (NH2 str as) and 3443cm -1 (NH2 str s) bands for 9methyladenine [24]. The absence of these bands in the IR spectrum of 1-methyladenine unambiguously shows the absence of amino groups in molecules of this compound isolated in argon. This is supported by the absence of the characteristic band of the NH2 bending vibration in the range 1650-1600 cm -1. Bands of this type of vibrations were observed in spectra of amine forms of adenine (the doublet 1644, 1636 cm -~) [24]. The spectrum of 1-methyladenine contains the intense doublet band at 3494, 3488 cm- 1. Calculation assigns it to the N H stretching vibration of the imidazole cycle. This assignment is corroborated by the frequencies of the N7(9)H stretching vibrations of some related compounds: the doublets 3491, 3487; 3480, 3477cm -~ for purine [24]; 3504 cm -~ for imidazole [25], and 3497, 3488 cm -~ for adenine [24]. The spectrum of 9-methyladenine does not include such a band [24] because the proton of the imidazole fragment is substituted by a methyl group. The splitting of the N7(9)H band of the imidazole fragment in the spectrum of 1-methyladenine may be due to the existence of Table 2. Relative energies (kcalmol-I), dipole moments (D) and ionization potentials (eV) of the 1-methyladenine tautomers calculated by the AM1 methoda

Erel /~ I1~'

I

lla

lib

III

0 3.16 8.43

0.79 4.07 8.56

5.13 3.13 8.62

5.44 9.29 8.60

~AM1 calculations give the planar structure of the ring in 1-methyladenine. bCalculated via Koopmans' theorem.

848

el al.

V. B. PIVOVAROV

0.5

1.2-

1.0

0.4

0.8.

0.3

o~ .t3 0.6 ..0 <

~

0.2' 0.4.

0.1 0.2

0 3500

3400

Wave number

o .~ <

'

3300

' 1600 '

1500

Wave number

( c m "1)

0.6

0.6-

0.5

0.5-

0.4

0.4-

0.3-

e~ <

1400

(cm -I)

0.3-

0.2-

0.1.

0 1400

0,1-

0 1300

1200

1100

Wave number (cm "1)

1000

1000

' ' ' ' ' ' ' I 800

' ~ ' ' 600

Wave number

Fig. 2. Infrared spectrum of 1-methyladenine in an argon matrix at

' ~ 400

( e r a "1)

12 K.

the two prototropic tautomers differing in the position of the proton in the imidazole cycle (structures I and IIa in Fig. 1). Still, it is not improbable that the splitting stems from the effect of the argon matrix [26] on the 1-methyladenine molecules. The weak experimental band at 3315cm -~ (Fig. 2) is assigned, on the basis of calculation (3307cm -l) to the N 1 0 - H stretching vibration of the iminogroup. The presence of this band in the experimental spectrum is corroborated by accumulation of the signal in this frequency range. Maybe, because of the low intensity of the NH stretching vibration of the iminogroup, it was not displayed in spectra of other compounds [17-21], which, perhaps, also are partially in an iminoform. However, similar bands in the spectra of the imine tautomers of 3-methyicytosine and 1-methylisocytosine were registered and have the values 3316 and 3353cm -I, respectively [10]. Besides, non-empirical calculations of the IR absorption spectrum of the imine tautomer of 5methylcytosine [18] yield 3319 cm-1 as the frequency of the NH stretching vibration of the iminogroup.

IR spectra of l-methyladenine in argon matrix and solutions

849

Table 3. Observed (T= 17 K, Ar matrix) and calculated IR spectra of l-methyladenine ~ Observed v

P

v

3494 3448 3315

0.43 sh 0.07

3490

Stg(100)

NgH str

A'

0.08 0.08 sh 1.25 sh sh 0.35 0.14 sh sh sh 1.02 0.19 0.11 0.11 0.08 0.06

S,2(100) Sis(92) Si7(90) $14(37),Sis(37), $13(26) St3(43), St4(26), SIs(26) Si3(33), S14(33), $15(33)

NtoH str CsH str C2H str CH3 str as CH 3 str as CH 3 str s

A' A' A'

2962 2929 1673 1666 1664 1630 1625 1601 1581 1577 1572 1563 1477 1445 1430 1423 1418

3314 3121 3037 2991 2976 2938 1662

Sn(35), $4(19), Sa(10)

CoNi0 str, C4C5 str

A'

1632 1593

$4(25),$2(22), Ss(18)

ring str ring str

A' A'

1570 1463 1451 1438

$8(22), $4(20), $6(20) S4t + $42 + $4~(95) S~(27), $3~ + $32(25)

ring str CHa bend ring str, CzH bend CH 3 bend

A' A' A' A"

1411

$4(22), St(21), $9(21)

C4C5 str, NtC2 str, C8C9 str

A'

1390 1384 1379 1371 1358 1348 1343 1338 1335 1324 1294 1279 1244 1223 1176 1135 1108 1088 1077 1067 1064 1053 1043 1039 958 921 826 818 802 697 649 619 571 565 560 555

sh sh 0.18 sh sh 0.48 sh sh sh sh 0.12 0.19 0.07 0.07 0.62 0.15 0.06 0.12 0.14 0.22 sh 0.20 0.14 0.05 0.22 0.06 sh 0.52 0.12 0.12 0.05 0.09 sh sh sh 0.44

~

Calculated PED

$2(28), Ss(24), $9(15)

$41-t-$42..I-$43(87)

Assignmenff

Symmetry

A" A' A'

1371

Ss(24), S,~ + $45(24)

ring str, CsH bend

A'

1354 1342

$41 + $42 + $43(92) S~+ $47(62), Sn(17)

CH3 bend N9H bend, C6N H str

A" A'

1340

$3(27), $7(23), S,~+ $47(13)

ring str, NgH bend

A'

1302 1276 1240 1207 1183 1143

S~ + $45(37), Ss(17) S31+ $32(34), $1(14) $13(35),$6(18), $2s(16) St(25), S31+ $32(21), $2(18) $37(55),S,A+S4s(24) Sa(19), $2t(19), $24(15)

CsH bend C2H bend NiCl2 str ring str, C2H bend N~oH bend, CsH bend ring str, ring bend

A' A' A' A' A' A'

1093

S3s+ $39 + $40(84)

CH3 bend, ring bend

A'

1070

S,(27), $3o(21), S~(15)

ring bend, ring str

A'

1057 1049 1022 964 935

S~(24), $2~(22), $21(17) S~ + $39 + $4o(72) S~(46), $5o(23), S~2(17) $20(23),S31+ $32(23) S~ + Sa9+ $4o(73)

ring bend, CsH bend CH3 bend, ring bend Call oop, C2H oop ring bend, C2H bend CHa bend, C2H bend

A' A' A" A' A"

823 788 705 638 610

$24(19),S21(19), S~(15) $5o(47),S~(19), $5.~(17) S,(26), S~(18), S~(14) S~ + S~(62), S24(19) S~(70), Ss5(19)

ring bend C2H oop, CsH oop ring bend CoNt() bend CsH oop, NgH oop

A' A" A' A' A"

564

$55(71),S~(21)

NgH oop

A"

continued overleaf

850

V. B. Pivovaaov et al. Table 3. continued

Observed v 532 526 519 512 454

Calculated Ih 0.20 0.04 0.23 0.09 0.05

v 541 527 523 502 446 387 375 348 320 217

PED $52(56),$55(17), $54(15) $57(22),$59(17), $51(16) $33(27),$35(24), $24(18) $58(23),$56(19), S~0(13) $62(32),$58(29) $63(24),$59(22), S01(14) $5~(67),S~(14), S~(ll) $48 + $49(71) Ssj(21), $65(19), .557(16) $53(68),$56(21)

Assignment"

Symmetry

NIoH oop ring oop, C6Ni0 oop ring bend ring oop ring oop ring oop C6N~0 oop NtC~2 bend ring oop NiCi2 oop

A" A"

A' A" A" A" A" A'

A" A"

° v, frequency in cm -~. °1, relative peak intensity; sh, band shoulder; braces indicate merged bands. c Abbreviations: as, antisymmetric; s, symmetric; str, stretching; bend, bending; oop, out-of-plane.

As the 1-methyladenine spectrum does not contain the aminogroup vibration bands at all, as mentioned above, when isolated in argon, molecules of 1-methyladenine only exist in the imine forms. Yet, analysis of the NH modes reveals the characteristic bands of the iminogroup in the spectrum. Weak absorption in the range 3300-3350 cm- ~is likely to be the spectral criterion of existence of imine tautomers, both for purine and for pyrimidine bases. The intensive band at 1176 cm -J corresponds to the plane bending vibrations of the N10-H bond (the calculated value is 1183 cm-t). C6=N10 and the ring modes The bands corresponding to the stretching vibrations of bonds formed by heavy atoms are situated in the range between 1700 and 1200cm -1 (Table 3). In the region below l l 0 0 c m -1 there are bands assigned to bending vibrations of the ring. The highest frequency band of the region (1666cm -~) belongs to the stretching vibration of the C6=N10 bond (calculation gives 1662 cm-~). It is the most intense band in the whole spectrum of 1-methyladenine. In the IR spectra of the aminoforms of adenine and 9methyladenine [24], in the region below 2000 cm -~, the highest-&equency bands are at 1644cm -~ (NH2 bending vibrations) and 1632cm -~ (ring stretching). Therefore, the band at 1666 cm -~ of the C6=N10 stretching vibration, as well as the aforementioned bands of the N10H vibrations (stretching and bending), can be used as the spectral indicator of the imine form of adenine and, possibly, of purines. Comparison of the bands of stretching vibrations of the ring of 1-methyladenine and adenine reveals essential differences in IR absorptions of the amine and imine structures. These differences take place both for the frequencies of certain vibrations and for potential energy distributions for vibrations of two different tautomer forms (in the case of similar frequencies). The reason for such differences is the unequal degrees of n-electron conjugation of the ring bonds. In adenine molecules studied earlier the degree of conjugation is higher, and therefore the electronic orders of bonds equalize and so do the force constants of the ring bonds. This results in pronounced mixing of contributions of several ring bonds into each particular vibration. In 1-methyladenine molecules, which exist in the iminoform, the degree of conjugation is lower, and thus some of the ring bonds have larger force constants and their vibrations do not mix with vibrations of other bonds so much. This is primarily true of the C4=C5, C2=N3 and N7=C8 bonds of 1-methyladenine. As a result, absorption bands in the IR spectrum of 1-methyladenine in the range 1630-1550 cm-~ are only of vibrations of the aforementioned bonds (Table 3). In the region below 1500 cm- J, bands of ring vibrations, as those for adenine, have more complicated structures and in many cases mix with the C-H and N-H bending vibrations of the C-H and N-H bonds.

IR spectra of 1-methyladenine in argon matrix and solutions

851

0.04 / ~

e

~ 0.02 <

0

~ 1700

1600 Wave number (cm "1)

1500

Fig. 3. IR spectra of solutions: (a) 1-methyladenine-d2 in D20, (b) 1-methyladenine-d2 in D20-1,4-dioxane mixture (volume percentage of dioxane is 90%); (c) 1-methyladenine-d 2 in CH3OD; (d) adenine-d3 in D20; (e) 1-methyladenosine-d2 in D20.

C-H modes The C-H vibrations are the least informative in terms of amino-imine tautomerism of 1-methyladenine. Therefore, in this section we restrict ourselves to the two remarks: (i) the frequencies of stretching and bending vibrations of the C-H bonds of methyl groups are observed in the usual well-known spectral regions. These vibrations practically do not mix with ring vibrations; (ii) the bands of the C2-H and C8-H stretching vibrations have never been observed in the exprimental spectra of purine bases isolated in inert matrices, though theoretically these vibrations are IR active. The results of nonempirical calculation of IR absorption band intensities available for some purine and pyrimidine bases also predict an extremely low intensity of C2H and C8H stretching vibrations.

Spectra of solutions In order to study the influence of the environment on the structure of 1-methyladenine molecules we investigated spectra of various solutions of this compound. 1-Methyladenine and its deuterated form (1-methyladenine-d2) were studied in CH3OH, CH3OD, D20, 1,4-dioxane and a water-dioxane mixture (CV2o= 10%; 50%). Spectral manifestations of tautomerism were studied for the wavelength range 1500-1750 cm -~, where there are bands carrying information on tautomerism (NH2 bending, C = N stretching) and also C = C stretching vibrations. The large width of the absorption bands which is characteristic of IR spectra of solutions results in large overlap of closely situated bands; therefore the absorption spectra of all the compounds studied consist of a few broad (with the halfwidth of about 10 cm- ~) bands (Fig. 3). The interpretation of the spectra of 1-methyladenine in solutions is based on the method of comparative analysis of the spectra of related compounds series which is common in practical spectroscopy. The presence of the iminoform of 1-methyladenine in an aqueous solution was revealed by the presence of the high frequency band at 1650 cm -~ with a considerable contribution of the C=N stretching vibration on the spectrum of the D20 solution. The assignment of this band is based on comparison of spectra of 1-methyladenine with those spectra of its analogues which are in the fixed imine (1-methyladenosine) and the dominating amine forms (adenine [27]). Also comparison of spectral alterations resulting from deuteration (Fig. 3 and Table 4) was used. Thus, the high frequency bands of

852

V. B. PIVOVAROV et al.

Tablc 4. Observed IR frcqucncies (cm -*) of l-methyladeninc in the solutionsand theircomparison with thosc of the related compounds

Compound

Solvent b

1-CH3-Ade-d2

D20 90% dxn CH3OD CH3OH D20 CH3OD CH3OH CH3OD CH3OH D20 CH3OD CH3OH D20 CHaOD 50% dxn 90% dxn

1-CH3-Ade

adenine-d3 adenine 9-CH3-Ade-d2 9-CH3-Ade 1-CH3-Ado-d2 1-CH3-Ado 2-NH2-Pyr-d2

dxn 2-NHz-Pyr 2-O-Pyr-d

4-O-Pyr-d

CH3OH D20 CH3OD 50% dxn 90% dxn D20 CH3OD

Wave numbeff -(1655 sh, -1685 --1655 -1660 (1648, 1650 1650 -------(1685 sh, (1680 w, 1674 (1658, (1668,

(1650, 1623 sh) 1643) 1643 ----1615 -1625 sh) --(1598, (1593, (1594, (1590, (1586, (1630, 1590) (1638, 1625 sh) 1650, 1640 sh) 1638, 1626 sh) (1640, 1626sh) 1632) 1632 w)

---1585 1630 1621 1600 -1600 w ---1567) 1560 m) 1560 m) 1554 m) 1552 m) 1563 1598 1605 1598 1600 1575 1580

1563 1559 1559 -1585 w 1576 w -1572 w 1556 w --1496 1496 1495 (1500 m, 1490) (1515 m, 1488) 1497 (1548, 1530 sh) (1545, 1540 sh) (1555 sh, 1544) 1532 1520 1516

"Abbreviations: 1-CH3-Ade, 1-methyladenine; 9-CH3-Ade, 9-methyladenine; 1-CH3-Ado, 1-methyladenosine; 2-NH2-Pyr, 2-aminopyrimidine; 2-O-Pyr, 2-oxopyrimidine; -d, -d 2, -d3 marks deuterated forms of the compounds. b dxn, 1,4-dioxane; 50% dxn, 90% dxn are the volume concentrations of 1,4-dioxane in D20. CThe letters following the frequencies represent intensities (m, medium; w, weak); sh, band shoulder. Parentheses indicate merged bands.

spectra of 1-methyladenine (as well as 1-methyladenine-d2) in the solutions studied are rather close in positions to those of 1-methyladenosine (Table 4), but essentially differ from those of the spectra corresponding to the solutions of adenine and adenine-d3 ( A v = 2 0 cm -I for D20 and A v = 22 cm -1 for CHaOD ). Deuteration of the compounds resulted in deuteration of the amino group, NH2---~ND2, and therefore in displacement of the NH2 bending band out of the range studied. Therefore, the number of bands in the spectra of solutions decreased after deuteration only in compounds containing an amino group instead of an imino group, which is corroborated by the shift of the 1655 cm-I band in the spectrum of adenine-d3 after deuteration (solution in CH3OD) and the 1630 cm -~ band in the spectrum of a solution of 2-aminopyrimidine-d2 in the same solvent. Similarly, the high-frequency band is not observed in the spectra of D20 solutions of adenine-d3 and 2-aminopyrimidine-d2. The spectra of the iminoforms of 1-methyladenosine and 1-methyladenine contain the high frequency bands, when solved in CH3OH (1650 and 1685cm-~), CH3OD (165 and 643cm-~), and D20 (1648 and 1643 cm-1). Thus, the 1643 cm -1 band in the spectrum of an aqueous (D20) solution of 1-methyladenine-d2 suggests the C = N stretching vibration, proves the presence of the imino group in the structure of its molecules and thus shows that the water solution contains at least a considerable fraction of imine tautomers.

CONCLUSION

The analysis of the IR spectrum of 1-methyladenine isolated in the low temperature Ar matrix shows total dominance of the iminoform in the frozen gas phase. The main reason for this conclusion is the presence of the characteristic imino group frequencies in the

IR spectra of 1-methyladenine in argon matrix and solutions

853

spectrum. The set of these experimentally revealed frequencies can be used as a spectral indicator of the presence of the "rare" iminoform of adenine and, possibly, of other nucleotide bases and their derivatives. In an aqueous solution, despite a considerable difference from the case of the matrix, the tautomer equilibrium is not shifted towards dominance of the 1-methyladenine aminoform. The result of a comparative analysis of spectra of 1-methyladenine and its related compounds solutions evidences the considerable content of the imine tautomer in water. Acknowledgements--This work has been partly supported by the Ukrainian State Committee of Science and Technology within the Project 2/381.

REFERENCES [1] S. Cradock and A. F. Hinchcliffe, Matrix Isolation. Cambridge University Press, Cambridge (1975). [2] G. G. Sheina, E. D. Radchenko and Yu. P. Blagoi, Dokl. A N SSSR 282, 1497 (1986). [3] E. D. Radchenko, A. M. Plokhotnichenko, A. Yu. Ivanov, G. G. Sheina and Yu. P. Blagoi, Biofizika 31, 373 (1986). [4] K. Szczepaniak and M. Szczesniak, J. Molec. Struct. 156, 29 (1987). [5] G. G. Sheina, S. G. Stepanian, E. D. Radchenko and Yu. P. Blagoi, J. Molec. Struct. 158, 275 (1987). [6] E. D. Radchenko, A. M. Plokhotnichenko, G. G. Sheina and Yu. P. Blagoi, Biofizika 28, 559 (1983). [7] E. D. Radchenko, G. G. Sheina, N. A. Smorygo and Yu. P. Blagoi, J. Molec. Struct. 116, 387 (1984). [8] K. Kuczera, M. Szczesniak and K. Szczepaniak, J. Molec. Struct. 172, 101 (1988). [9] M. J. Nowak, L. Lapinski and J. Fulara, Spectrochim. Acta 45A, 229 (1989). [10] M. Szczesniak, M. J. Nowak and K. Szczepaniak, J. Molec. Struct. 115, 221 (1984). [11] S. G. Stepanian, G. G. Sheina, E. D. Radchenko and Yu. P. Blagoi, Zh. Fiz. Khim. 63, 3008 (1989). [12] S. G. Stepanian, E. D. Radchenko, G. G. Sheina and Yu. P. Blagoi, J. Molec. Struct. 216, 77 (1990). [13] M. J. Nowak, K. Szczepaniak, A. Barski and D. Shugar, J. Molec. Struct. 62, 49 (1980). [14] D. Shugar and K. Szczepaniak, Int. J. Quant. Chem. 20, 373 (1981). [15] M. J. Nowak, J. Fulara and L. Lapinski, J. Molec. Struct. 175, 91 (1988). [16] L. Lapinski, J. Fulara and M. J. Nowak, Spectrochim. Acta 46A, 61 (1990). [17] M. J. Nowak, L. Lapinski and J. Fulara, Spectrochim. Acta 45A, 229 (1989). [18] L. Lapinski, M. J. Nowak, J. Fulara, A. Les and L. Adamowicz, J. Phys. Chem. 94, 6555 (1990). [19] M. Szczesniak, Ph.D. Thesis, Institute of Physics, Polish Academy of Sciences, Warsaw (1985). [20] M. Szczesniak, J. Leszczynski and W. B. Person, J. Am. Chem. Soc. 114, 2731 (1992). [21] M. Szczesniak, K. Szczepaniak, J. S. Kwiatkowski, K. KuBulat and W. B. Person, J. Am. Chem. Soc. ll0, 8319 (1988). [22] M. J. S. Dewar, E. G. Zoebisch, E. F. Healy and J. J. P. Stewart, J. Am. Chem. Soc. 107, 3902 (1985). [23] L. A. Gribov and V. A. Dementiev, Methods and Algorithms o f Computation in the Theory o f Vibrational Spectra o f Molecules (in Russian), p. 356. Nauka, Moscow (1981). [24] S. G. Stepanian, G. G. Sheina, E. D. Radchenko and Yu. P. Blagoi, J. Molec. Struct. 131,333 (1985). [25] S. T. King, J. Phys. Chem. 74, 2133 (1970). [26] M. J. Nowak, L. Lapinski, J. S. Kwiatkowski and J. Leszczynski, Spectrochim. Acta 47A, 87 (1991). [27] M. Tsuboi, S. Takahashi and I. Harada, Physico-chemical Properties o f Nucleic Acids (Edited by J. Duchesne), Vol. 2. Academic Press, London (1973).

~(A) st:s-H